Polarization immunoassay

Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

The determination of theophylline in plasma can also be accomplished by various immunoassay techniques.66-67 Theophylline was also determined by a polarization fluoroimmunoassays but found to have a caffeine interference.88. In a more research oriented application, the interaction of caffeine with L-tryptophan was studied using h NMR with the results indicating that caffeine interacted with tryptophan in a 1 1 molar ratio through parallel stacking.69... 

Tibi, L. and Burnett, D., Interference by caffeine in polarization immunoassays for theophylline, Ann. Clin. Biochem., 291 1994. 

Guo XQ, Castellano FN, Li L, Lakowicz JR (1998) Use of a long lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular weight analytes. Anal Chem 70 632-637... 

This is used for synthesis of porphobilinogen (Eq. 10.24).25 Porphobilinogen is the key building block in the biosynthesis of pigments of life such as porphyrins, heme, and vitamin B12. Interesting application of porphobiliogen to synthesis of immunocomponents for the measurement of lead (Pb) by fluorescence polarization immunoassay has been reported.26... 

Most of the reported methods of analysis of valproic acid and its sodium salt in biological fluids rely on the use of chromatography, especially gas chromatography, although high performance liquid chromatography (HPLC) is also reported. Other methods, such as flow injection analysis, enzyme-immunoassay, fluorescence-polarization capillary electrophoresis, and potentiometry are sometimes used. The reported methods can be classified as follows. 

A competitive fluorescence-polarization immunoassay method was described for the monitoring of 12 drugs including valproic acid [18]. Samples (serum or plasma) were deproteinated. Fluorescence from the fluorescein-labeled analyte used as tracer was excited at 488 nm and polarization of light emitted at 531 nm was measured. The calibration was stable for 4 weeks and the coefficient of variation was below 10%. A single measurement took 8-10 min. 

Five anticonvulsants including valproic acid were determined by the Abbott TD x fluorescence polarization immunoassay automatic analyzer. Recoveries were 94.8-106% and the coefficients of variations were 1.0-9.7% [23], Fluorescence polarization immunoassay and enzyme immunoassay were compared for the determination of free valproic acid in serum [24], Good correlation (R = 0.9992) was obtained between the two assays. Higgins [25] reported on the determination of valproic acid in serum by enzyme immunoassay with use of EMIT reagents and the Abbot ABA-200 analyzer. Responses were rectilinear up to 150 mg/L. 

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

A.Y. Kolosova, J.H. Park, S.A. Eremin, S.J. Kang and D.H. Chung, Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl. J. Agric. Food Chem. 51, 1107-1114 (2003). 

Immunoassay is the sterile measurement of a drug molecule as an antigen using a specific antibody. Detection is performed by UV light absorption, radioactivity, or fluorescence polarization. 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

Rao, M.L. et al. 1994. Monitoring tricyclic antidepressant concentrations in serum by fluorescence polarization immunoassay compared with gas chromatography and HPLC. Clin Chem. 40 929. 

BMP-IA bacterial magnetic particle-based immunoassay FIIA flow-injection immunoassay PFIA polarization fluoroimmunoassay. b BDD12AC benzyldimethyldodecylammonium chloride DDAC didecyldimethylammonium chloride. 

Fluorescence microscopy, 16 483 Fluorescence polarization (FP), 14 149-150 Fluorescence polarization immunoassay (FPIA), 12 97... 

Immunoassays based on fluorescence polarization have become very popular because, in contrast to radioimmunoassays, they require no steps to separate free and bound tracer. In a fluoroimmunoassay, the fluorescently labeled antigen is... 

Other successful homogeneous assays have been developed such as the fluorescence polarization technique made available by Abbott Laboratories of North Chicago, USA. This competitive immunoassay technique relies upon the principle... 

Figure 14.5. Representation of fluorescence polarization immunoassay measurements, (a) Unbound tracer (b) bound tracer.

Since FPIAs are conducted as homogeneous immunoassays, they are susceptible to effects from endogenous fluorophores and from intersample variations. Such problems and others due to the sample matrix are largely avoided by sample dilutions of several hundredfold. Low-affinity, nonspecific binding of tracers to sample proteins, when present in sufficiently high concentrations, can result in a falsely elevated polarization signal. Interference from sample proteins can be eliminated when warranted, by proteolytic hydrolysis with pepsin.(46)... 

W. B. Dandliker, R. J. Kelly, J. Dandliker, J. Farquhar, and J. Levin, Fluorescence polarization immunoassay. Theory and experimental method, Immunochemistry 10, 219-227 (1973). 

P. Urios, N. Cittanova, and M.-F. Jayle, Immunoassay of the human chorionic gonadotropin using fluorescence polarization, FEES Lett. 94, 54-58 (1978). 

P. Urios and N. Cittanova, Adaptation of fluorescence polarization immunoassay to the assay of macromolecules, Anal. Biochem. 185, 308-312(1990). 

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